Project Summary Telomerase is the ribonucleoprotein complex capable of extending human telomeres, preventing replicative senescence and allowing for cellular immortality. Expression of hTERT, the catalytic subunit of telomerase, is actively transcribed in the inner cell mass during development, when organismal telomere length is established, and telomerase remains on in adult stem cell compartments later. Additionally, over 90% of cancers reactivate telomerase to survive telomere crisis. However, the process that mediates the activation of telomerase activity in development and oncogenesis still largely unknown. The process of cellular reprogramming by the Yamanaka factors (Oct4/Klf4/Sox2/cMyc) recapitulates the activation of hTERT transcription and of telomerase activity, and as such, is a robust system for studying multiple processes behind telomerase induction in development and disease. It is crucial to understand the similarities and differences between telomerase reactivation in pluripotent and cancer cells to better understand this key developmental process and inform the development of more efficient targeted cancer therapies. The complexity of the extended hTERT promoter led us to comprehensively search for regulatory elements in the surrounding genome that govern transcription from the hTERT promoter. To pursue this avenue of inquiry, we performed a STARR-seq screen for hTERT regulatory sequences and identified a number of putative enhancers and repressors in the locus surrounding hTERT. Furthermore, alternative splicing of hTERT provides another regulatory layer to hTERT transcription, modulating hTERT transcription through nonsense-mediated degradation of variant transcripts. We identified the alternative splice variant, hTERT ?Ex2, the expression of which is strongly inversely correlated with telomerase activity in stem, somatic, and cancer cells. This proposal aims to i) identify and functionally validate cis-regulatory elements affecting hTERT transcription in stem and cancer cells, and ii) elucidate the role of hTERT exon-2 exclusion in regulation of telomerase activity. These studies will provide valuable insight into the transcriptional mechanisms that govern telomerase activity and potentially provide novel therapeutic modalities for the treatment of cancer.